Project Summary Glioblastoma (GBM) is the most prevalent and malignant primary brain tumor. Despite aggressive multimodal chemo- and radiation therapy, median survival remains less than two years. Over the last years, it has become clear that glioma-specific, self-renewing cancer stem cells [Glioma Stem Cells (GSC)] are critical for initiation and therapeutic resistance of GBM. Therapeutic targeting of GSCs is thus considered a promising avenue to treat GBM. Here, we propose to directly examine cellular translation in GSCs. This critical aspect of gene regulation is poorly understood in GSCs. Yet, deregulation of translation is associated with oncogenesis in other tumors. We will use ribosome profiling to examine GSCs and differentiated glioma cells (DGCs) from patient-derived xenografts (PDXs). Preliminary data demonstrate the feasibility of this approach and show that protein production in GSCs is controlled to a significant degree at the level of translation. Based on these data, we hypothesize that translational control is a major determinant of the stem-like state of GSCs, and that ribosome profiling of PDXs can guide the identification of novel, GSC-specifc therapeutic targets. Our proposal aims to globally identify translationally regulated genes in GSCs and to delineate regulatory nodes that determine protein production in GSCs. We will utilize this insight to identify novel, GSC-specific therapeutic targets for GBM treatment, using shRNA on a panel of identified genes. The proposed work is expected to provide new insight into GSC biology, and identify new GSC-specific therapeutic targets. We anticipate obtained data to guide precision medicine strategies for GBM treatment.